UBC12 is one of 50+ members of the human ubiquitin conjugating enzyme (E2) family that covalently transfers the ubiquitin-like protein NEDD8 onto proteins. Its major substrates are cullins which function in the context of multi-subunit enzymes known as cullin-RING ubiquitin ligases (CRLs). NEDD8 modification activates CRLs to stimulate their ability to synthesize ubiquitin polymers on their bound protein substrates. CRLs account for ~20% of all cellular protein degradation by the proteasome and alterations in regulatory mechanisms controlled by CRLs are associated with diseases such as cancer and neurodegerative disorders, inflammation and immune response modulation, and viral and bacterial infections. As a result of their important functions in protein homeostasis and direct impact on human health, CRLs and associated regulatory mechanisms have received considerable attention for developing human therapeutics. The overall goal of this application is to screen a large compound library from the MLPCN to identify modulators of UBC12 and prioritize these inhibitors in secondary and tertiary assays devised to categorize the potency, selectivity, and cell permeability of primary hits. Our approach employs a TR-FRET biochemical assay we have developed and validated that reconstitutes the transfer of NEDD8 from UBC12 onto a specific lysine residue of the cullin CUL1. Primary hits from our screen will be prioritized based on known chemical features and in secondary and tertiary assays designed to examine UBC12 selectivity, cell permeability, and potency on cancer cells. These will inform subsequent SAR to improve on-target effects both in vitro and on treated cells. Probes emerging from our study are expected to yield novel tools for detailed biochemical and structural studies on UBC12 as well as provide unprecedented insight into the regulation of cullin neddylation and CRLs. We anticipate these efforts will stimulate the development of E2 selective probes to elucidate the functions and regulation of ubiquitin and ubiquitin-like protein modification systems, leading to the validation of these enzymes as a therapeutic target class to directly benefit human health.